WO2019096115A1 - Récepteur de lymphocytes t isolé, cellule modifiée par celui-ci, acides nucléiques codants, vecteur d'expression, procédé de préparation, composition pharmaceutique et applications - Google Patents

Récepteur de lymphocytes t isolé, cellule modifiée par celui-ci, acides nucléiques codants, vecteur d'expression, procédé de préparation, composition pharmaceutique et applications Download PDF

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WO2019096115A1
WO2019096115A1 PCT/CN2018/115191 CN2018115191W WO2019096115A1 WO 2019096115 A1 WO2019096115 A1 WO 2019096115A1 CN 2018115191 W CN2018115191 W CN 2018115191W WO 2019096115 A1 WO2019096115 A1 WO 2019096115A1
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cell
her2
cells
tcr
chain
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侯亚非
侯大炜
谭贤魁
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杭州康万达医药科技有限公司
合成免疫股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention is in the field of biotechnology, and in particular, relates to an isolated T cell receptor, a modified cell thereof, a nucleic acid encoding, an expression vector, a preparation method, a pharmaceutical composition, and an application.
  • Her2/neu is a transmembrane protein belonging to the EGFR family, which forms dimers with other family proteins and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signaling pathways (see literature “Growth Factors,” 2008;26:263", “Oncol Biol. Phys, 2004; 58: 903”).
  • Her2/neu protein is overexpressed in a variety of epithelial-derived cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc.
  • Herceptin targeting Her2/neu can significantly prolong the survival of Her2/neu-positive breast cancer patients (see the literature "N Engl J Med, 2001, 344: 783”), whereas Herceptin alone is used to treat Her2 positive
  • the clinical response rate for metastatic breast cancer is only 11% to 26% (see the literature "J Clin Oncol, 2002; 20: 7169”), indicating that Heceptin alone is not ideal for most Her2 high-expressing metastatic breast cancer.
  • Heceptin combined with chemotherapy increases clinical response rates, most Her2/neu overexpressing breast cancer patients develop resistance to Heceptin after one year (see the literature "J Clin Oncol, 2001; 19: 2587”).
  • Tumor vaccines targeting the Her2/neu 369-377 polypeptide antigen enter clinical trials, although clinical phase 1/2 shows that this vaccine can induce specific T killer cells against the Her2/neu 369-377 polypeptide antigen (see literature "Breast Care” , 2016; 11:116"), but the clinical phase III did not achieve the intended goal of extending patient survival (http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast- Cancer-halted-after-futility-review).
  • CARs chimeric antigen receptors
  • T-cell therapies adoptive transfer of in vitro cultured, chimeric antigen receptors (CARs)-based tumor-specific T-cell therapies, developed as the first CAR-T targeting Her2/neu antigens for solid tumors
  • CRS cytokine release syndrome
  • Severe cytokine storms and neurotoxicity are common toxicities in CAR-T therapy (see the literature “Blood, 2016; 127: 3321”), partly due to the unrestricted CAR, a non-native T cell receptor.
  • Cell activation is involved (see the literature "Nat Ned, 2015; 21: 581”) or with autocrine cytokine that does not require antigen stimulation (see the literature "Cancer Immunol Res, 2015; 3: 356”).
  • TCR-T therapy for adoptive transfer of T cells transfected with specific T cell receptor (ie TCR) genes is considered to be the most promising immune cell gene therapy for solid tumors (see the literature “Adv Immunol. 2016; 130:279 -94”).
  • clinical studies of TCR-T therapy targeting NY-ESO-1 antigen have shown encouraging clinical therapeutic effects (see the literature “Nat Med. 2015 Aug; 21(8): 914-921”).
  • the number of specific TCRs currently known to target tumor antigens and to efficiently recognize tumor cells is very limited, thus limiting the widespread use of TCR-T therapies.
  • TCR-T therapy does not exhibit the severe cytokine storm toxicity exhibited by CAR-T therapy
  • target antigen if the target antigen is derived from a self-protein, targeting a target antigen that is low in normal tissue cells may cause serious Autoimmune response, ie on target off tumor toxicity (Blood 2009; 114: 535-546).
  • a general strategy is to perform point mutations in the complementarity determing regions (CDRs) on the TCR in vitro, or by never undergoing a central tolerance mechanism. Induction was performed in a screened humanized mouse T cell pool (see the literature "Front Immunol. 2013; 4:363").
  • the present invention provides isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions, and uses.
  • the present invention provides:
  • An isolated T cell receptor comprising at least one of an ⁇ chain and a ⁇ chain, the ⁇ chain and the ⁇ chain each comprising a variable region and a constant region, characterized in that the T cell receptor
  • the antigen Her2/neu expressed by the tumor cell can be specifically recognized, and the amino acid sequence of the variable region of the ⁇ chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 19, the ⁇ The amino acid sequence of the variable region of the strand has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • T cell receptor (2) The T cell receptor according to (1), wherein the T cell receptor is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule; Yes, the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  • An isolated nucleic acid encoding a T cell receptor comprising a coding sequence of at least one of an ⁇ chain and a ⁇ chain of said T cell receptor, said ⁇ chain coding sequence and said ⁇ chain coding sequence Including a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing an antigen Her2/neu expressed by a tumor cell, and the amino acid sequence encoded by the ⁇ chain variable region coding sequence has At least 98% identity to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence encoded by the ⁇ chain variable region coding sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • nucleic acid according to (6) wherein the nucleic acid is DNA or RNA.
  • the T cell receptor encoded by the nucleic acid is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule;
  • the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  • nucleic acid according to any one of (6) to (11), wherein the ⁇ chain coding sequence and the ⁇ chain coding sequence are linked by a coding sequence of a cleavable linker polypeptide.
  • a recombinant expression vector comprising the nucleic acid according to any one of (6) to (14), and/or a complement thereof, which is operably linked to a promoter.
  • a T cell receptor-modified cell the surface of which is modified by the T cell receptor according to any one of (1) to (5), wherein the cell comprises a primitive T cell or a precursor thereof Cells, NKT cells, or T cell lines.
  • the method of transfection comprises: transfection with a viral vector, preferably, the viral vector comprises a ⁇ retroviral vector or a lentiviral vector;
  • the chemical means comprises a method of transfection with a liposome; physically, preferably, the physical means comprises an electrotransfection mode.
  • a pharmaceutical composition comprising the T cell receptor-modified cell according to (16) as an active ingredient, and a pharmaceutically acceptable excipient.
  • composition according to (24), wherein the pharmaceutical composition comprises the T cell having a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course per patient. Receptor modified cells.
  • composition according to (24), wherein the pharmaceutical composition is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular and intraperitoneal administration.
  • a method of treating a tumor and/or a cancer comprising administering a T cell receptor-modified cell according to (16) to a tumor and/or a cancer patient.
  • the invention has the following advantages and positive effects:
  • the present invention successfully induces T cell clones specific for HLA-A2-presented Her2/neu epitope polypeptides (such as Her2/neu 369-377 polypeptide) from HLA-A2-positive healthy donor peripheral blood.
  • a T cell clone carrying a native TCR that specifically recognizes a Her2/neu epitope polypeptide (such as Her2/neu 369-377 polypeptide) is screened therefrom, and the entire TCR sequence is obtained.
  • This TCR belongs to CD8 molecule-dependent, has a moderate affinity for Her2/neu epitope polypeptides (such as Her2/neu 369-377 polypeptide), and specifically recognizes HLA-A2 positive and expresses Her2/neu antigen-bearing tumor cells.
  • T cell clones carrying this TCR were screened by the central immune tolerance mechanism and entered the peripheral T cell bank.
  • the killer T cells carrying this TCR were present in normal human peripheral blood and did not cross-react to normal tissue cells that express Her2/neu protein in a small amount.
  • the present invention achieves a novel TCR that is capable of specifically recognizing tumor antigens while avoiding possible off-target toxic side effects.
  • the constant region of the TCR is also engineered (eg, by disulfide modification or murine transformation) to further reduce or avoid mismatching with the endogenous TCR when expressed on immune cells. occur.
  • Immunological cells modified with this TCR can specifically recognize a variety of HLA-A2 + and Her2/neu + tumor cell lines with significant anti-tumor effect.
  • the TCR used to modify immune cells does not cross-react to normal cells that express Her2/neu in a small amount. Therefore, TCR-T therapy based on this TCR is expected to treat a variety of solid tumors.
  • the TCR-modified immune cells of the invention provide a new choice for treating HLA-A2 + and Her2/neu + tumor patients, and have good industrial application prospects.
  • FIG. 1 shows a table of Her2/neu 369-377 polypeptide (Her2-E75)-specific killer T cells induced from HLA-A2 + normal donor PBMC (specifically #2 PBMC) in Example 1 of the present invention.
  • Figure 1A shows the results of flow cytometry analysis of PBMC cells stained with CD8-APC antibody and Her2-E75 pentamer-PE after two rounds of Her2-E75 antigen polypeptide stimulation.
  • the right panel is stimulated by peptides.
  • the CD8 + pentamer + killer T cell population was subjected to FACS sorting to obtain T cell clones.
  • the left panel shows control cells without polypeptide stimulation.
  • FIG. 1B shows the phenotypic analysis of flow cytometry of CD8 + E75-tetramer + killer T cell clones stained with CD8-APC and Her2-E75 tetramer-PE.
  • the right panel shows CD8 + Her2 tetramer + T cell clone
  • Her2 CTL 1B5 is a purified Her2-E75 polypeptide-specific CTL cell clone.
  • the left panel shows control CTL cells without polypeptide stimulation.
  • the abscissa indicates the fluorescence intensity of CD8 molecule expression, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • Figure 1C shows the results of T cell clonal function assay.
  • T cell clone Her2 CTL 1B5 slanted bar graph
  • control CTL cells without peptide stimulation point bar graph
  • the cell supernatant was taken for ELISA analysis of IFN- ⁇ , and each test group and control group were duplicated, and the results were shown as mean ⁇ SD.
  • the abscissa indicates the different experimental groups, and the ordinate indicates the concentration of T cells secreting IFN- ⁇ .
  • Figure 2 shows two constructed lentiviral vectors carrying the Her2 TCR-1B5 TCR gene (shown as "pCDH-EF1 ⁇ -Her2 TCR-(PGK-GFP) vector” and "pCDH-EF1 ⁇ -Her2 TCR vector, respectively"
  • the main functional fragment The fragment shown above simultaneously expresses the TCR gene driven by the EF-1 ⁇ promoter and the GFP gene driven by the PKG promoter, and the fragment shown below expresses only the TCR gene.
  • the beta and alpha chains of each TCR are linked by the coding sequence of the cleavable linker polypeptide (furin-F2A).
  • FIG. 3 shows the results of phenotypic and functional assays of T cell lines transfected with the Her2 TCR-1B5 TCR gene.
  • J. RT3-T3.5 T cell line J. RT3
  • the GFP + Her2-E75 tetramer + cell population shown in Figure 3A is a cell expressing Her2 TCR-1B5 TCR, expressed as a percentage of each positive cell population to the total number of cells.
  • the TCR involved in the left panel adds a disulfide bond structure (Her2 TCR-1B5-dis) to the constant region of the ⁇ chain and the ⁇ chain
  • the TCR involved in the right panel is the constant region of the human ⁇ chain and ⁇ chain by the mouse constant region.
  • the homologous sequence was replaced (Her2 TCR-1B5-mC).
  • the abscissa indicates the fluorescence intensity of GFP molecule expression
  • the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • Figure 3B shows the expression of the constant regions of the TCR alpha and beta chains in different T cell variants after modification in different ways.
  • Her2 TCR-1B5-dis refers to a TCR in which the constant regions of the ⁇ chain and the ⁇ chain each add a disulfide bond structure;
  • Her2 TCR-1B5-mC means that the constant regions of the human ⁇ chain and the ⁇ chain are small.
  • the TCR replaced by the homologous sequence of the murine constant region.
  • the GFP + Her2-E75 tetramer + cells are positive cells expressing Her2 TCR-1B5 TCR, and the ordinate is the percentage of the TCR-positive cells to total GFP + cells.
  • the abscissa indicates different T cell line groups, wherein "Jurkat(TCR a+b+)" refers to Jurkat cells, both of which express both the ⁇ chain and the ⁇ chain, and "J.RT3(TCR a+b-)" refers to J .RT3-T3.5 cells, which are derived from Jurkat cells, and the ⁇ chain gene is deleted, and the ⁇ chain is still expressed.
  • Figure 3C shows that a T cell strain transfected with a lentiviral vector encoding the Her2 TCR-1B5 TCR gene can recognize the Her2-E75 polypeptide presented by T2 cells.
  • TCR alpha chain and the beta chain were modified in different ways, and TCR cells expressing TCR were mixed with T2 cells presented with different concentrations of Her2-E75 polypeptide for 16 hours, stained with anti-CD69-PE antibody, and subjected to flow cytometry analysis.
  • J.RT3-Her2-1B5-dis indicates J.RT3-T3.5 cells expressing Her2 TCR-1B5-dis
  • J.RT3-Her2-1B5-mC indicates expression of Her2 TCR-1B5-mC J.RT3-T3.5 cells
  • Jurkat-Her2-1B5-dis indicates Jurkat cells expressing Her2 TCR-1B5-dis
  • Jurkat-Her2-1B5-mC indicates Jurkat expressing Her2 TCR-1B5-mC cell.
  • the abscissa indicates the concentration of the Her2-E75 polypeptide presented by the T2 cells.
  • the ordinate is the percentage of CD69 + cells to total GFP + cells.
  • Figure 4 shows the phenotypic and functional test results of peripheral blood mononuclear cells (PBMC) transfected with the Her2 TCR-1B5-mC TCR gene.
  • Figure 4A shows the results of flow cytometric analysis of PBMCs from two different donors transfected with Her2 TCR-1B5-mC, stained with Her2-E75 tetramer-PE and anti-CD8-APC antibody. First, the lymphocyte population was separated according to the morphology and size of the cells, and the Her2-E75 tetramer + cell population was a cell expressing Her2 TCR-1B5 TCR.
  • the abscissa indicates the fluorescence intensity of CD8 molecule expression
  • the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • the percentages shown are the ratio of the number of lymphocytes to each positive cell population.
  • the left panel relates to a peripheral blood mononuclear cell (#1PBMC) provided by a donor, and the right panel relates to a different donor-provided PBMC (#2 PBMC).
  • CD8 + Her2-E75 tetramer + cells are killer T cells expressing Her2 TCR-1B5-mC.
  • the CD8 - Her2-E75 tetramer + cells may be CD4 + helper T cells expressing Her2 TCR-1B5-mC.
  • FIG. 4B shows that T cells expressing Her2 TCR-1B5-mC can recognize the Her2-E75 polypeptide presented by T2 cells.
  • Two different donor PBMCs transfected with the lentiviral vector encoding Her2 TCR-1B5-mC and GFP were mixed with T2 cells with different concentrations of Her2-E75 polypeptide for 16 hours, and the cell supernatant was taken for IFN- ⁇ .
  • ELISA analysis The target cells in the control group were T2 cells which presented the EBV virus antigen polypeptide LMP2 426-434 which binds to the HLA-A2 molecule.
  • T2+Her2-E75 0.1 ⁇ g/ml indicates a T2 cell group in which 0.1 ⁇ g/ml of Her2-E75 polypeptide was presented
  • T2+Her2-E75 0.01 ⁇ g/ml indicates that Her2 was present at 0.01 ⁇ g/ml.
  • - T75 cell group of E75 polypeptide indicates a T2 cell group of 0.001 ⁇ g/ml of Her2-E75 polypeptide
  • T2+EBV-LMP 1 ⁇ g/ml indicates that 1 ⁇ g was presented.
  • FIG. 4C shows the results of the CD8 antibody blocking assay for T cell function.
  • the #2 PBMC transfected with the lentiviral vector encoding Her2 TCR-1B5-mC and GFP gene was co-cultured with the antigen polypeptide presented by T2 cells, and anti-human CD8 antibody was added to detect whether the function of T cell secreting IFN- ⁇ was detected. suppressed.
  • T2+Her2-E75 0.1 ⁇ g/ml indicates a T2 cell group in which 0.1 ⁇ g/ml of Her2-E75 polypeptide was added without anti-human CD8 antibody
  • T2+Her2-E75 0.1 ⁇ g/ml+anti -CD8 indicates a T2 cell group in which 0.1 ⁇ g/ml of Her2-E75 polypeptide was added to the anti-human CD8 antibody.
  • the abscissa indicates the different experimental groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • Each of the test groups and the control group in Figs. 4B and 4C was a duplicate well, and the results were shown as mean ⁇ SD.
  • FIG. 5 shows the results of functional assays of peripheral blood mononuclear fine (PBMC)-recognizing tumor cell lines transfected with the Her2 TCR-1B5-mC TCR gene.
  • Fig. 5A shows that the lentiviral vector encoding the Her2 TCR-1B5-mC TCR gene was transfected with #2 PBMC, and after mixing with different tumor cell lines for 16 hours, the cell supernatant was taken for ELISA analysis of IFN- ⁇ .
  • Each test group and control group were duplicate wells, and the results were shown as mean ⁇ SD.
  • the abscissa indicates the different experimental groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • Figure 5B shows the addition of anti-CD8 antibody (shown as “colo205+anti-CD8”) or anti-HLA-ABC antibody to the culture well of the transfected #2 PBMC-targeted Colo205 cells, respectively (shown as "colo205+anti-HLA-ABC”), the function of the function blocking test results.
  • the abscissa indicates the different tumor cell line groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by the T cells.
  • Colo205 and Coca-2 are HLA-A2 positive Her2/neu positive colon cancer cells
  • MAD-MB-231 is HLA-A2 positive Her2/neu positive breast cancer cells
  • H647 is HLA-A2 negative Her2/neu positive lung cancer cells
  • SK-OV-3 is an HLA-A2 negative Her2/neu positive ovarian cancer cell
  • Bjab is an HLA-A2 positive Her2/neu negative lymphoma cell.
  • the Her2/neu antigen belongs to the tumor-associated antigen, and the high-affinity T cells that recognize the Her2/neu antigen are mostly cleared by the central tolerance mechanism to avoid possible autoimmune responses (see the literature "Immunol Rev. 2016; 271(1) ): 127-40"). Therefore, it has become very difficult to induce a T cell clone having a Her2/neu antigen specifically recognized by a tumor cell from a peripheral blood T cell pool.
  • the Her2/neu 369-377 polypeptide naturally derived from the cell may be glycosylated to cause a difference in TCR recognition configuration (see the literature "Proc. Natl. Acad. Sci. USA 2003; 100: 15029 - 15034" ).
  • high-affinity T cell clones that can only recognize exogenously loaded antigenic peptides often obtain dominant expansion, and can specifically recognize the cell-producing
  • the growth of T cell clones of the endogenous Her2/neu antigen polypeptide is inhibited (see the literature "J Exp Med.
  • Screening for a high-affinity TCR T cells transfected with this high-affinity TCR recognize multiple tumor cells of HLA-A2 + Her2/neu + (see the literature "HUMAN GENE THERAPY 2014; 25: 730-739" ).
  • This TCR was not obtained from monoclonal T cells and therefore it was not possible to determine whether this TCR is a native TCR present in a T cell bank that has been screened for central tolerance.
  • Her2/neu protein is also expressed in small organs such as myocardium, lung, esophagus, kidney, and bladder (see “Oncogene. 1990 Jul; 5(7): 953-62"), so it is based on high affinity Her2/.
  • TCR-T therapy with neu antigen-specific TCR has a risk of off-target toxicity to normal tissues.
  • the tumor cells have high expression of Her2/neu protein, so the number of antigenic peptides presented by HLA on the cell surface will increase accordingly.
  • the difference in the number of HLA/antigen polypeptide complexes between tumor cells and normal cells can become normal T cells. And the window of the tumor tissue.
  • the present invention proposes to obtain the sequence of the native TCR from the auto-T cell repertoire, and then to express the TCR on the T cell in vitro, so that the obtained TCR-expressing T cells can recognize the increased expression of the tumor cells.
  • Her2/neu antigen is the key to the successful development of effective and low toxicity TCR-T therapy.
  • the present invention induces Her2/neu 369-377 presented to HLA-A2 from HLA-A2-positive healthy donor peripheral blood.
  • the polypeptide has a specific T cell clone and is screened for a T cell clone carrying a native TCR having a moderate affinity for the Her2/neu 369-377 polypeptide.
  • the present invention recognizes that specific T cell clones directed against the Her2/neu 369-377 polypeptide will proliferate after immunization with the Her2/neu 369-377 antigen polypeptide and thus cannot represent the natural presence of the in vivo T cell bank (repertoire).
  • a specific T cell population that recognizes the Her2/neu 369-377 polypeptide antigen presented by the target cell is a specific T cell population that recognizes the Her2/neu 369-377 polypeptide antigen presented by the target cell.
  • the present invention also does not employ a method by which other research groups induce polypeptide-specific T cells from HLA-A2 negative peripheral blood (see the document "The Journal of Immunology, 2010, 184: 1617 - 1629"), although it is easier to obtain from allogeneic PBMCs.
  • High-affinity allo-T cells recognizing the Her2/neu 369-377 polypeptide antigen are obtained, but this also increases the allogeneic response caused by T cell cross-recognition of other polypeptides presented by HLA-A2 molecules.
  • the present invention provides an isolated T cell receptor comprising at least one of an alpha chain and a beta chain, both of which comprise a variable region and a constant region, characterized in that The T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by the tumor cell, and the amino acid sequence of the variable region of the ⁇ chain has at least 98%, preferably the amino acid sequence shown in SEQ ID NO: At least 98.5%, more preferably at least 99% identity, the amino acid sequence of the variable region of the beta chain having at least 98%, preferably at least 98.5%, more preferably at least the amino acid sequence set forth in SEQ ID NO: 99% consistency as long as it does not significantly affect the effects of the present invention. It is also preferred that the amino acid sequence of the variable region of the ⁇ chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the variable region of the ⁇ chain is set forth in SEQ ID NO: 20.
  • variable regions of the TCR alpha and beta chains are used to bind the antigenic polypeptide/major histocompatibility complex (MHC I), respectively comprising three hypervariable regions or complementarity determining regions (CDRs), ie, CDR1, CDR2, CDR3.
  • the CDR3 region is critical for the specific recognition of antigenic peptides presented by MHC molecules.
  • the TCR alpha chain is composed of different V and J gene segments, and the ⁇ chain is composed of different V, D and J gene segments.
  • the MHC class I molecule includes human HLA.
  • the HLA includes: HLA-A, B, C.
  • the T cell receptor is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule.
  • the amino acid sequence of the antigen Her2/neu is shown in SEQ ID NO: 17.
  • the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  • the HLA-A2 alleles expressed by HLA-A2 positive cells include HLA-A*0201, 0202, 0203, 0204, 0205, 0206, and 0207.
  • the HLA-A2 molecule is preferably HLA-A*0201.
  • the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 369-377 polypeptide (SEQ ID NO: 18). In other embodiments, the antigenic epitope polypeptide of the antigen Her2/neu is 4-9 consecutive identical amino acids to the Her2/neu 369-377 polypeptide (eg, 4, 5, 6, 7, 8, or 9) Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-11 amino acids in length. For example, in one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 373-382 polypeptide (SEQ ID NO: 25).
  • the T cell receptor recognizes a maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide between 1.0 and 10 ng/ml (eg, between 3.0 and 8.0 ng/ml, 5.0 to 7.0 ng/ml) ). In one embodiment of the invention, the maximum half-reactive polypeptide concentration is about 6.9 ng/ml.
  • maximum half-reactive polypeptide concentration refers to the concentration of the desired polypeptide that induces a T cell response to reach a maximum of 50%.
  • the maximum half-reactive polypeptide concentration of specific T cells against the cytomegalovirus (CMV) antigen CMV pp65 (495-503) polypeptide is reported to be between 0.1-1 ng/ml, and this TCR is considered to be high for CMV antigen polypeptides. Affinity (see the literature "Journal of Immunogical Methds 2007; 320: 119-131").
  • the T cell receptor has a moderate affinity for the Her2/neu antigen, thereby avoiding off-target toxicity which may be caused by high affinity.
  • the exogenous TCR ⁇ chain and ⁇ chain expressed by T cells may be mismatched with the ⁇ chain and ⁇ chain of TCR itself, not only diluting the expression level of the correct paired exogenous TCR, but also the antigen specificity of the mismatched TCR. It is clear that there is a potential danger of recognizing the autoantigen, and therefore it is preferred to modify the constant regions of the TCR alpha and beta chains to reduce or avoid mismatches.
  • the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the invention finds that the constant region of the alpha chain may be wholly or partially The ground is replaced by a homologous sequence derived from other species, and/or the constant region of the beta strand may be replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse.
  • the replacement can increase the expression level of TCR in the cells, and can further increase the specificity of the cells modified by the TCR to the Her2/neu antigen.
  • the constant region of the alpha chain may be modified with one or more disulfide bonds, and/or the constant region of the beta chain may be modified with one or more disulfide bonds, such as one or two.
  • TCR is modified in two different ways.
  • Her2 TCR-1B5-dis is a disulfide bond added to the TCR constant region by point mutation in the literature "Cancer Res. 2007 Apr 15; 67 ( 8): 38.9-903., which is incorporated herein by reference in its entirety.
  • Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety by reference. This article.
  • amino acid sequence of the alpha chain is set forth in SEQ ID NOs: 2, 6, or 10
  • amino acid sequence of the beta strand is set forth in SEQ ID NOs: 4, 8, or 12.
  • the sequence is the original human sequence; for the amino acid sequence such as the ⁇ chain shown in SEQ ID NO: 6, it has one in the constant region. Sulfur bond; for the ⁇ chain of the amino acid sequence such as SEQ ID NO: 10, the constant region is replaced with a murine constant region.
  • the sequence is the original human sequence; for the ⁇ chain of the amino acid sequence such as SEQ ID NO: 8, it has a modification in the constant region. Sulfur bond; for the ⁇ chain of the amino acid sequence such as SEQ ID NO: 12, the constant region is replaced with a murine constant region.
  • the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 2, and the amino acid sequence of the beta strand is set forth in SEQ ID NO: 4.
  • the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 6
  • the amino acid sequence of the beta strand is set forth in SEQ ID NO: 8.
  • the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 10
  • the amino acid sequence of the beta strand is set forth in SEQ ID NO: 12.
  • the alpha chain of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence set forth in SEQ ID NOs: 2, 6, or 10.
  • the alpha chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs: 2, 6, or 10.
  • the beta strand of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence set forth in SEQ ID NOs: 4, 8, or 12.
  • the ⁇ chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs: 4, 8 or 12.
  • the alpha and/or beta strands of the TCRs of the invention may also bind to other functional sequences at the terminus (e.g., the C-terminus), such as the functional region sequences of the costimulatory signals CD28, 4-1BB, and/or CD3zeta.
  • the invention also provides an isolated T cell receptor-encoding nucleic acid comprising a coding sequence for at least one of an alpha chain and a beta chain of said T cell receptor, said alpha chain coding sequence and beta strand coding
  • the sequences each comprise a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by the tumor cell, and the amino acid encoded by the ⁇ chain variable region coding sequence
  • the sequence has at least 98%, preferably at least 98.5%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence encoded by the ⁇ chain variable region coding sequence having SEQ ID NO:
  • the amino acid sequence shown at 20 has a consistency of at least 98%, preferably at least 98.5%, more preferably at least 99%, as long as the effects of the present invention are not significantly affected. It is also preferred that the ⁇ chain variable region coding sequence encodes the amino acid sequence set forth
  • the nucleic acid can be DNA or RNA.
  • the alpha chain variable region coding sequence is set forth in SEQ ID NO: 21, and the beta chain variable region coding sequence is set forth in SEQ ID NO: 22.
  • the T cell receptor encoded by the nucleic acid is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule.
  • the epitope polypeptide of the antigen Her2/neu is a Her2/neu 369-377 polypeptide (SEQ ID NO: 18).
  • the antigenic epitope polypeptide of the antigen Her2/neu is 4-9 consecutive identical amino acids to the Her2/neu 369-377 polypeptide (eg, 4, 5, 6, 7, 8, or 9) Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-10 amino acids in length.
  • the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 373-382 polypeptide (SEQ ID NO: 25).
  • the T cell receptor encoded by the nucleic acid recognizes a maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide between 1.0 and 10 ng/ml (eg, between 3.0 and 8.0 ng/ml, 5.0- Between 7.0ng/ml). In one embodiment of the invention, the maximum half-reactive polypeptide concentration is about 6.9 ng/ml. In this case, the T cell receptor has a moderate affinity for the Her2/neu antigen, and the off-target toxicity which may be caused by high affinity can be avoided.
  • the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the alpha chain constant region coding sequence is derived in whole or in part from other
  • the homologous sequence of the species is replaced, and/or the beta strand constant region coding sequence is replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse.
  • the replacement can increase the expression level of TCR in the cells, and can further increase the specificity of the cells modified by the TCR to the Her2/neu antigen.
  • the alpha chain constant region coding sequence may comprise a coding sequence for one or more disulfide bonds, and/or the beta chain constant region coding sequence may comprise a coding sequence for one or more disulfide bonds.
  • the alpha chain coding sequence is set forth in SEQ ID NOs: 1, 5 or 9
  • the beta strand coding sequence is set forth in SEQ ID NOs: 3, 7 or 11.
  • the sequence is the original human sequence; and for the ⁇ chain of the coding sequence such as SEQ ID NO: 5, it has one in the constant region. Sulfur bond; for the ⁇ chain of the coding sequence as shown in SEQ ID NO: 9, the constant region is replaced with a murine constant region.
  • the sequence is the original human sequence; for the ⁇ chain of the coding sequence such as SEQ ID NO: 7, it has a modification in the constant region. Sulfur bond; for the ⁇ chain of the coding sequence as shown in SEQ ID NO: 11, the constant region is replaced with a murine constant region.
  • the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 1, and the coding sequence for the beta chain is set forth in SEQ ID NO: 3.
  • the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 5
  • the coding sequence for the beta chain is set forth in SEQ ID NO: 7.
  • the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 9, and the coding sequence for the beta chain is set forth in SEQ ID NO:11.
  • the alpha chain coding sequence and the beta strand coding sequence are joined by a coding sequence for a cleavable linker polypeptide, which increases the expression of the TCR in the cell.
  • cleavable linker polypeptide means that the polypeptide functions as a linker and can be cleaved by a specific enzyme, or the nucleic acid sequence encoding the polypeptide is translated by ribosome skipping so that it is ligated The polypeptides are separated from one another.
  • cleavable linker polypeptides examples include F2A polypeptides, including but not limited to F2A polypeptides from picornaviruses, and class 2A sequences similar to other viruses.
  • the cleavable linker polypeptide also includes a standard canonical four amino acid motif, i.e., the R-X-[KR]-R amino acid sequence, which can be cleaved by the Furin enzyme.
  • the TCR encoded by this embodiment is a single-stranded chimeric T cell receptor, and after the expression of the single-stranded chimeric T cell receptor is completed, the cleavable linker polypeptide linking the ⁇ chain and the ⁇ chain is cleaved by a specific enzyme in the cell. Thereby forming equal amounts of free alpha and beta chains.
  • alpha and beta chains constituting the single-stranded chimeric TCR can also be replaced, as described above, in whole or in part by homologous sequences derived from other species, and/or modified (coding) one or more disulfide bonds.
  • sequence of the nucleic acid is set forth in SEQ ID NOs: 13, 15, or 23.
  • the nucleotide sequence of the nucleic acid is encoded sub-optimized to increase gene expression, protein translation efficiency, and protein expression, thereby enhancing the ability of the TCR to recognize antigen.
  • Encoding sub-optimization includes, but is not limited to, modification of the translation initiation region, alteration of mRNA structural fragments, and use of different codons encoding the same amino acid.
  • the sequence of the above TCR-encoding nucleic acid can be mutated, including the removal, insertion and/or substitution of one or more amino acid codons such that the expressed TCR recognizes the function of the Her2/neu antigen unchanged or enhanced.
  • conservative amino acid substitutions are made comprising substitution of one amino acid in the variable region of the TCR alpha chain and/or beta chain described above with another amino acid having similar structural and/or chemical properties.
  • similar amino acid refers to an amino acid residue having similar properties of polarity, electrical load, solubility, hydrophobicity, hydrophilicity, and the like.
  • the mutated TCR still has the biological activity of recognizing the above-described Her2/neu antigen polypeptide presented by the target cell.
  • the TCR maturation modification is performed, ie, comprising amino acids of the complementarity determining region 2 (CDR2) and/or CDR3 regions of the variable regions of the TCR alpha chain and/or beta chain described above. Removal, insertion and/or substitution alter the affinity of the TCR to bind to the Her2/neu antigen.
  • the invention also provides an isolated mRNA transcribed from the DNA according to the invention.
  • the invention also provides a recombinant expression vector comprising a nucleic acid (e.g., DNA) according to the invention operably linked to a promoter, and/or a complement thereof.
  • a nucleic acid e.g., DNA
  • the DNA of the invention is suitably operably linked to a promoter, enhancer, terminator and/or polyA signal sequence.
  • the combination of the above-described functional elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA.
  • the basic backbone of the recombinant expression vector can be any known expression vector, including plasmids or viruses, including but not limited to, for example, retroviral vectors (the virus prototype is Moloney murine leukemia virus (MMLV)) and lentiviruses. Vector (the virus prototype is human immunodeficiency type I virus (HIV)).
  • retroviral vectors the virus prototype is Moloney murine leukemia virus (MMLV)
  • Vector the virus prototype is human immunodeficiency type I virus (HIV)
  • a recombinant vector expressing a TCR of the present invention can be obtained by recombinant DNA techniques conventional in the art.
  • expression of the alpha and beta chain genes on the recombinant expression vector can be driven by two different promoters, including various known types, such as strongly expressed, weakly expressed, Inducible, tissue-specific, and differentiation-specific promoters.
  • the promoter may be of viral or non-viral origin, such as the CMV promoter, the promoter on the LTR of MSCV, the EF1-alpha promoter, and the PGK-1 promoter.
  • the driving directions of the two promoters can be either in the same direction or in the opposite direction.
  • expression of the alpha and beta chain genes on the recombinant expression vector can be driven by the same promoter, such as in the case of a single chain chimeric T cell receptor, the nucleotide sequence of the alpha chain and The nucleotide sequence of the beta strand is ligated by the Furin-F2A polypeptide coding sequence.
  • the recombinant expression vector may comprise coding sequences for other functional molecules in addition to the alpha and beta chain genes.
  • an autofluorescent protein such as GFP or other fluorescent protein
  • Another embodiment includes expressing an inducible suicide gene system, such as inducing expression of a herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducing expression of a Caspase 9 (iCasp9) protein. Expression of these "safety-switches” can increase the safety of the cells modified by the TCR genes of the present invention for use in vivo.
  • HSV-TK herpes simplex virus-thymidine kinase
  • iCasp9 Caspase 9
  • Another embodiment includes expressing a human CD8 gene, comprising expressing the CD8 alpha chain and the beta chain, either alone or in combination, and the cells modified by the TCR gene of the present invention can enhance their ability to specifically recognize the Her2/neu antigen by expressing the CD8 molecule, or CD8-negative T cells (such as CD4 + T helper cells) acquire the ability to specifically recognize the Her2/neu antigen.
  • a human chemokine receptor gene such as CCR2, which binds to a corresponding chemokine ligand that is highly expressed in tumor tissue, thereby enhancing cells modified by the TCR gene of the present invention Homing in tumor tissue.
  • the present invention also provides a T cell receptor-modified cell whose surface is modified by a T cell receptor according to the present invention, wherein the cell comprises a primitive T cell or a precursor cell thereof, NKT cell, or T Cell line.
  • Modification in the "T cell receptor modification” means that a cell expresses a T cell receptor of the present invention by gene transfection, that is, the T cell receptor is anchored through a transmembrane region.
  • the modified cells are on the cell membrane and have the function of recognizing the antigenic polypeptide/MHC complex.
  • the invention also provides a method of preparing a T cell receptor modified cell according to the invention, comprising the steps of:
  • the cells of step 1) can be derived from mammals, including humans, dogs, mice, rats and their transgenic animals.
  • the cells can be derived from autologous or allogeneic. Allogeneic cells include cells from identical twins, allogeneic stem cells, genetically engineered allogeneic T cells.
  • the cells of step 1) include naive T cells or their precursor cells, NKT cells, or T cell strains.
  • naive T cell refers to mature T cells in peripheral blood that have not been activated by the corresponding antigen.
  • T cells can be isolated by methods known in the art.
  • T cells can be obtained from different tissues and organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, cord blood, and tumor tissue.
  • the T cells can be obtained from hematopoietic stem cells (HSCs), including bone marrow, peripheral blood, or cord blood, isolated by stem cell marker molecules such as CD34.
  • HSCs hematopoietic stem cells
  • the T cells can be derived from inducible pluripotent stem cells (iPS cells), including introducing a specific gene or a specific gene product into the somatic cells, transforming the somatic cells into stem cells, and inducing differentiation into T cells or precursors thereof in vitro. cell.
  • T cells can be obtained by a usual method such as density gradient centrifugation, and examples of density gradient centrifugation include Ficoll or Percoll density centrifugation.
  • One embodiment is the production of enriched T cells from peripheral blood using plasma apheresis or leukapheresis.
  • One embodiment is a method of separating a magnetic cell by labeling a specific cell population with an antibody (eg, Enriched CD8 + or CD4 + T cells were obtained by system (Miltenyi Biotec), or by flow cytometry.
  • the T cell precursor cells are hematopoietic stem cells.
  • the gene encoding the TCR of the present invention can be directly introduced into hematopoietic stem cells, and then transferred to the body to further differentiate into mature T cells; the coding gene can also be introduced into T cells which are differentiated and matured by hematopoietic stem cells under specific conditions in vitro.
  • the cells can be resuspended in a cryopreservation solution and stored in liquid nitrogen.
  • cryopreservation solutions include, but are not limited to, 10% (v/v) DMSO and 90% (v/v) human serum or fetal bovine serum.
  • the cells were frozen at -80 ° C at a temperature of 1 ° C per minute and then stored in the gas phase portion of the liquid nitrogen tank.
  • Other cryopreservation methods are to freeze the cells placed in the cryopreservation solution directly into -80 ° C or liquid nitrogen.
  • the nucleic acid of step 2) is a nucleic acid according to the invention, including the DNA and RNA.
  • the transfection includes physical, biological, and chemical means.
  • the physical method is to introduce the TCR gene into the cell in the form of DNA or RNA by calcium phosphate precipitation, liposome, microinjection, electroporation, gene gun, and the like.
  • Commercial instruments are currently available, including electrotransfers (eg Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830 (BTX) (Harvard Instruments, USA), Gene Pulser II (BioRad, USA), Multiporator (Eppendort, Germany)
  • the biological method is to introduce the TCR gene into a cell through a DNA or RNA vector, and a retroviral vector (such as a ⁇ retroviral vector) is a common tool for transfecting and inserting a foreign gene fragment into an animal cell (including a human cell).
  • viral vectors are derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses, etc.
  • the chemical method is to introduce polynucleotides into cells, including colloidal dispersion systems, such as macromolecular complexes, nanocapsules, Microspheres, microbeads, micelles, and liposomes.
  • TCR gene is introduced into a cell
  • various assays are used to analyze whether the gene of interest is introduced into the target cell, including common molecular biology methods ( For example, Southern and Northern blots, RT-PCR and PCR, etc., or common biochemical methods (eg ELISA and Western) Trace), and methods of the present invention mentioned.
  • the transfection is carried out by a retroviral vector or a lentiviral vector.
  • T cells can be expanded in vitro by co-activation of a TCR/CD3 complex on the surface and a co-stimulatory molecule (such as CD28).
  • Stimulators that activate TCR, CD3, and CD28 can be adsorbed on the surface of a culture vessel, or on the surface of a co-culture (such as a magnetic bead), or can be directly cultured in a cell culture medium.
  • Another embodiment is to co-culture T cells with trophoblast cells that express helper stimulatory molecules or corresponding ligands including, but not limited to, HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • helper stimulatory molecules or corresponding ligands including, but not limited to, HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • the T cell culture is cultured and expanded under appropriate culture conditions according to the usual method of in vitro culture of mammalian cells. For example, when the cells reach more than 70% confluence, they can be passaged, usually 2 to 3 days for fresh culture. When the cells reach a certain number, they are used directly or frozen as described above. The in vitro culture time can be within 24 hours or as long as 14 days or longer. After the frozen cells are thawed, the next step can be applied.
  • the cells can be cultured in vitro for hours to 14 days, or any number of hours in between.
  • T cell culture conditions include the use of basal culture fluids including, but not limited to, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo.
  • Conditions required for survival and proliferation of other cells include, but are not limited to, the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF- ⁇ and TNF-a, other culture additives (including amino acids, sodium pyruvate, vitamin C, 2-mercaptoethanol, growth hormone, growth factors) .
  • the cells can be placed under suitable culture conditions, for example, the temperature can be at 37 ° C, 32 ° C, 30 ° C or room temperature, and the air conditions can be, for example, air containing 5% CO 2 .
  • the invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
  • the tumor and/or cancer is antigenic Her2/neu positive and is HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
  • the invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for detecting a tumor and/or cancer of a host.
  • a sample of a tumor and/or a cancer cell taken out from a host can be contacted with a T cell receptor-modified cell of the present invention at a concentration, and can be judged according to the degree of reaction between the two. Whether the tumor and/or cancer is HLA-A2 positive or HLA-A2 negative, and whether the antigen Her2/neu is expressed.
  • the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises, as an active ingredient, a T cell receptor-modified cell according to the present invention, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition preferably comprises the T cell receptor modified cells in a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient, including any between the two endpoints The number of cells.
  • each course of treatment is 1-3 days, administered 1-3 times a day.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the pharmaceutically acceptable excipients include pharmaceutically or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives, including sugars, lipids, polypeptides, amino acids, antioxidants, adjuvants, Preservatives, etc.
  • the pharmaceutical composition can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, and peritoneal Internal administration.
  • the invention also provides a method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient a T cell receptor modified cell according to the invention.
  • the tumor and/or cancer is antigenic Her2/neu positive and is HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
  • the T cell receptor modified cells are preferably administered at a dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient.
  • each course of treatment is 1-3 days, administered 1-3 times a day.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the T cell receptor-modified cells can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular and intraperitoneal administration.
  • the T cell receptor-modified cells can eliminate the tumor cells expressing the Her2/neu antigen and/or alter the microenvironment of the tumor tissue to induce other anti-tumor immune responses after entering the therapeutic subject.
  • the present invention also provides the use of the isolated T cell receptor for detecting proliferation or survival of the TCR-T cell in a patient treated with the TCR-modified T cell (ie, TCR-T cell), thereby performing Drug metabolism studies, and understanding the efficacy and toxicity of this TCR-T cell.
  • the TCR sequence can be used as a primer to detect the number of TCR-T cells carrying the TCR in vivo by a PCR method. Compared to methods in which fluorescently labeled HLA/polypeptide complex multimers are stained and analyzed by flow cytometry, the amount of cells required for such applications is less and more sensitive.
  • the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
  • the cell line used to prepare the lentiviral particles was 293T cells (ATCC CRL-3216).
  • the T cell strains used to detect TCR phenotype and function were Jurkat cells (clone E6-1, ATCC TIB-152) and J. RT3-T3.5 cells (ATCC TIB-153).
  • the presenting cell strain used to present the antigenic polypeptide was T2 cells (174xCEM.T2, ATCC CRL-1992).
  • the tumor cell lines for detecting TCR function are human colon cancer colo205 cells (ATCC CCL-222) and Caco-2 cells (ATCC HTB-37), human breast cancer MDA-MB-231 cells (ATCC HTB-26), human ovary Cancer SK-OV-3 cells (ATCC HTB-77), human lymphoma cells Bjab (ACC-757-DSMZ), human lung cancer H647 cells (ATCC CRL-5824) and H1355 cells (ATCC CRL-5865).
  • the cell line was maintained in RPMI-1640 complete medium (Lonza, cat#12-115F), and 10% calf serum FBS (ATCC 30-2020) was added to RPMI-1640 complete medium, 2mmol/L L-glutamic acid. , 100 ⁇ g/ml penicillin and 100 ⁇ g/ml streptomycin.
  • Peripheral blood products from healthy donors used in the trial were from the Pacific Blood Center in San Francisco (#1PBMC and #2 PBMC are Trima Residual Cell Components #R32334 and #R33941 from the Apheresis Collection Kit, respectively).
  • CTL Her2/neu 369-377-specific killer T cells
  • Peripheral blood was centrifuged ( ⁇ 400 g) by Ficoll-Paque Premium (Sigma-Alorich, cat# GE-17-5442-02) for 30 minutes.
  • Mononuclear cells PBMC
  • HLA-A2 phenotype of the cells was detected by fluorescein FITC-labeled anti-HLA-A2 antibody (Biolegend, cat#343303).
  • the RNA of the positive cells was extracted, reverse-transcribed into cDNA and cloned into the vector. Then, HLA gene sequencing analysis was performed to determine the cell matching type as HLA-A*0201.
  • the HLA-A2-positive PBMC cells were cultured in a culture well of a 24-well culture plate, and the culture solution was the above RPMI-1640 complete medium. 2 ⁇ 10e6/ml PBMC per well was added with Her2/neu 369-377 polypeptide (Her2-E75, synthesized with Peptide 2.0, 10 ⁇ g/ml dissolved in DMSO) at a concentration of 1 ⁇ g/ml.
  • cytokines were added, human IL-2 (Peprtech, cat#200-02) 100 IU/ml, human IL-7 (Peprotech) , cat#200-07) 5ng/ml, human IL-15 (Peprotech, cat#200-15) 5ng/ml.
  • 10 to 14 days of culture, antigen re-stimulation of cultured T cells 10e6 culture cells were added to each well in a 24-well plate, and 2 ⁇ 10e6 25 ⁇ g/ml mitomycin C (Santa Cruz Biotechnology) was added.
  • the T cell phenotype expressing the Her2/neu 369-377 specific TCR was analyzed by flow cytometry.
  • the cells to be tested were collected in 1.5 ml tubes (the number of cells was approximately 10e5) using 1 ml of DPBS solution (2.7 mM KCl, 1.5 mM KH 2 PO 4 , 136.9 mM NaCl, 8.9 mM Na 2 HPO 4 ⁇ 7H 2 O, pH 7.4) Wash once and reset in 100 ⁇ l of DPBS containing 1% calf serum, add 5 ⁇ l of fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat #300912), and 10 ⁇ l of fluorescein PE-labeled Her2- E75/HLA-A2 tetramer (Her2-E75 tetramer, MBL International Co, cat#T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer,
  • the flow cytometer was MACSQuant Analyzer 10 (Miltenyi Biotec), and the result analysis was performed using Flowjo software (Flowjo Corporation). T cell clones were obtained by single cell separation using a flow cytometer (FACS sorter). PBMCs stimulated with Her2/neu369-377 polypeptide antigen were stained with APC-labeled anti-human CD8 antibody and PE-labeled Her2-E75/HLA-A2 pentamer, followed by flow cytometry (Model: Sony cell sorter SH800) .
  • HLA-A2 positive autologous was treated by treatment with 25 ⁇ g/ml mitomycin C for 2 hours.
  • PBMC cells 10e5 cells per well, were incubated with 1 ⁇ g/ml Her2/neu 369-377 polypeptide overnight, and then added RPMI-1640 containing IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml. Complete medium.
  • the culture medium containing the cytokine was replaced every 3-4 days, and it was observed under the microscope whether or not T cell clones grew.
  • the proliferating T cells are collected, subjected to antigen re-stimulation as described above to obtain a sufficient number of cells, subjected to phenotypic or functional detection, and extracted RNA for cloning of the TCR gene.
  • T cell function assay The function of the T cell strain modified by Her2/neu 369-377 polypeptide-specific TCR is detected by the expression of CD69 on the surface of T cells. 10 e5 TCR cells transfected with TCR gene and 10e5 T2 cells were added to each well of a 96-well plate, and mixed culture was carried out in 100 ⁇ l/well of RPMI-1640 complete medium, and each test group was a duplicate well.
  • Her2/neu 369- The 377 polypeptide was then cultured overnight in an incubator at 5% CO 2 at 37 °C. The cells were collected and suspended in DPBS + 1% FBS solution, stained with APC-labeled anti-human CD69 antibody, and subjected to flow cytometry analysis. T cells expressing CD69 after stimulation with Her2/neu 369-377 antigen are thought to carry and express a TCR specific for Her2/neu 369-377.
  • Her2/neu 369-377-specific CTL clone and the function of primary T cells transfected with the TCR gene in PBMC were determined by detecting gamma interferon secreted in the supernatant of the cell after antigen stimulation.
  • Her2/neu 369-377-specific CTL clones or PBMC cells transfected with TCR gene were mixed with target cells in 96-well plates in 100 ⁇ l/well RPMI-1640 complete medium, and the target cells were added 10 times.
  • RNA from T cell clones using the Zymo Quick-RNA Microprep kit (Zymo Research, cat#R1050), using this as a template to obtain cDNA using the Smarter RACE 5'/3' kit (Takara, USA) Bio, cat#634858).
  • PCR was carried out using 5'-CDS primer and TCR ⁇ chain 3' primer 5'-GCCTCTGGAATCCTTTCTCTTG-3' (SEQ ID NO: 26) and ⁇ chain 3' primer 5'-TCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 27) TCR ⁇ and ⁇ full sequence gene fragments were added and cloned into pRACE vector (Takara Bio, cat#634858, USA). The competent bacteria Stellar (Takara Bio, cat #636763, USA) was transformed and plasmids were obtained and sequenced.
  • the viral vector for expression of TCR is a replication-deficient lentiviral vector, including: GFP-expressing lentiviral vector pCDH-EF1 ⁇ -MCS-(PGK-GFP), available from System Biosciences (Cat# CD811A-1); and the vector pCDH-EF1 ⁇ -MCS which does not express GFP, was obtained by removing the PGK promoter and GFP gene on the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector by a conventional technique in the art.
  • GFP-expressing lentiviral vector pCDH-EF1 ⁇ -MCS-(PGK-GFP) available from System Biosciences (Cat# CD811A-1)
  • the vector pCDH-EF1 ⁇ -MCS which does not express GFP was obtained by removing the PGK promoter and GFP gene on the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector by a conventional technique in the art.
  • the TCR ⁇ chain and the ⁇ chain and the entire gene sequence of the cleavable F2A sequence and the Furin fragment are synthesized, and linked to the multiple cloning site downstream of the EF-1 ⁇ promoter of the vector
  • the transcription sequence of the inserted TCR is TCR ⁇ chain (no stop codon), Furin fragment, F2A fragment, TCR ⁇ chain (see the literature “Gene Ther. 2008 Nov; 15(21): 1411–1423”).
  • the vector expressing GFP is driven by the inverted PGK promoter.
  • the vector that does not express GFP removes the PGK promoter as well as the GFP fragment.
  • TCR lentiviral particles were obtained by transfecting 293T cells with Lipofectaine 3000 transfection reagent (Thermo Fisher, cat# L3000001). Prepare 293T cells and transfection procedures according to the manufacturer's instructions. The transfection was carried out in a 96-well culture plate. First, a liposome solution of the transfected plasmid was prepared using Opti-MEM 1 culture solution (Thermo Fisher Co., cat #51985091), and P3000 reagent (P3000 reagent) was added to 250 ⁇ l of the culture solution according to the manufacturer's instructions.
  • Lipofectaine 3000 transfection reagent Thermo Fisher, cat# L3000001
  • P3000 reagent P3000 reagent
  • the TCR lentiviral vector plasmid and the viral packaging plasmid of the pCDH system SBI, cat#LV500A-1
  • another 250 ⁇ l of the culture solution was added with Lipofectaine 3000 reagent, and mixed for 15 minutes, and then added to the 293T cell culture well.
  • the cells were cultured for 16 hours at 37 ° C under 5% CO 2 , and changed to DMEM medium (Thermo Fisher Company, cat #11965092) containing 10% FBS. After 24 hours and 48 hours, the cell supernatants were collected and centrifuged at 2000 g.
  • the virus particles obtained by filtration with a 0.4 ⁇ m filter membrane were used to infect cells.
  • Recombinant TCR lentivirus was transfected into human T cells: cryopreserved primary PBMC cells were thawed and cultured in RPMI-1640 complete medium for 24 hours, and the dead cells were removed by Ficoll-Paque Premium density gradient centrifugation ( ⁇ 400 g) for 30 minutes.
  • Example 1 Induction of Her2/neu 369-377 polypeptide-specific killer T cells from HLA-A2 positive normal donor peripheral blood
  • polypeptide-specific killer T cells were induced from normal PBMC (#2 PBMC) by two rounds of in vitro stimulation with a low concentration of Her2/neu 369-377 polypeptide at 1 ⁇ g/ml, and flow cytometry and single cell separation were performed. .
  • the specific method is as described above. The results are as follows:
  • the right panel of Figure 1A shows that 0.013% of lymphocytes are CD8-positive killer T cells that bind to Her2/neu 369-377/HLA-A2 pentamer (ie, Her2-E75 pentamer), and there is no Her2 polypeptide in the left panel.
  • the stimulated control cells did not show CD8 positive pentamer positive cells.
  • the results indicate that the number of specific T cells recognizing the Her2/neu 369-377 antigen polypeptide is small in the natural T cell pool. Despite the small number, this group of T cells recognizing the Her2/neu 369-377 polypeptide can still be clearly distinguished.
  • high-affinity T cells and low-affinity T cells are further contained in the positive cells.
  • 453 CD8-positive pentameric positive cells were isolated by flow cytometry and subjected to monoclonal culture. After two rounds of antigen polypeptide re-stimulation and cytokine amplification, only one proliferation was obtained from the 453 isolated single T cells.
  • T cell clone Her2 CTL clone 1B5 (referred to as Her2 CTL 1B5).
  • FIG. 1B right panel shows that this purified CD8 + CTL clone binds to the Her2/neu 369-377/HLA-A2 tetramer (i.e., Her2-E75 tetramer).
  • Her2/neu 369-377 antigen polypeptide presented by 5x10 3 T2 cells (Her2/neu 369-377 antigen polypeptide was 10-fold diluted from 1 ⁇ g/ml to obtain After mixed cultures at a final concentration of 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, and 0.001 ⁇ g/ml, the IFN- ⁇ secreted by T cells in the supernatant was detected to determine the specificity of the T cell clone.
  • FIG. 1C shows that T cell clone 1B5 can be activated by a minimum concentration of 1 ng/ml of antigenic polypeptide, and the recognition function of T cells is positively correlated with the concentration of antigenic polypeptide (dose-dependent), indicating the specificity of this T cell clone.
  • the Her2/neu 369-377 polypeptide presented by HLA-A2 was identified. More importantly, this T cell clone also recognized a colon cancer cell line colo205 of HLA-A2 + Her2/neu + . Therefore, this T cell clone Her2 TCR-1B5 not only recognizes the Her-E75 polypeptide, but also activates by colo205 cells to secrete IFN- ⁇ .
  • This example obtained directly from the Her2/neu 369-377 polypeptide-specific CTL clone obtained in Example 1 containing ⁇ and ⁇ chains which are matched (i.e., the two strands can together constitute a functional TCR of the recognition antigen polypeptide).
  • the complete TCR gene sequence, which encodes a TCR is called "Her2 TCR-1B5".
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 2
  • the coding sequence is shown in SEQ ID NO: 1
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 4
  • the coding sequence is SEQ. ID NO: 3 is shown.
  • This TCR is present in the peripheral T cell pool of HLA-A2 positive normal humans and does not cross-react to normal cells that express Her2/neu protein in a small amount, resulting in an autoimmune response.
  • the specific detection method is as described above. The results are as follows:
  • FIG. 2 shows a schematic representation of the constructed TCR lentiviral vector structural fragment.
  • the alpha and beta chains are linked by a resectable furin recognition fragment and an F2A polypeptide fragment.
  • the lentiviral vector shown as "pCDH-EF1 ⁇ -Her2 TCR-(PGK-GFP) vector
  • PGK-GFP reverse PGK promoter Driven
  • Another expression vector (shown as "pCDH-EF1[alpha]-Her2 TCR vector") (bottom of Figure 2) removes GFP and its promoter sequences. By reducing the length of the vector to increase the production of lentiviral particles, the interaction between the two promoters is avoided, thereby increasing the expression of TCR.
  • the following sequences were ligated to the above-described GFP-expressing vector and the GFP-expressing vector, respectively: i) a nucleotide sequence of a TCR ⁇ -chain and an ⁇ -chain which are linked by a cleavable linker polypeptide and which have a disulfide bond added in the constant region ( SEQ ID NO: 13) (corresponding TCR is Her2 TCR-1B5-dis, amino acid sequence is set forth in SEQ ID NO: 14); ii) constant region linked by a cleavable linker polypeptide is replaced by a human sequence to a murine The nucleotide sequence of the TCR ⁇ chain and the ⁇ chain of the source sequence (SEQ ID NO: 15) (corresponding TCR is Her2 TCR-1B5-mC, amino acid sequence is shown as SEQ ID NO: 16); iii) by cleavability Linking the nucleotide sequence of the original TCR ⁇ chain and the ⁇ chain linked by the polypeptid
  • Her2 TCR-1B5-dis recombinant lentiviral vector (carrying GFP);
  • Her2 TCR-1B5-mC recombinant lentiviral vector (carrying GFP);
  • Her2 TCR-1B5-mC w/o GFP recombinant lentiviral vector without carrying GFP.
  • Her2 TCR-1B5-wt recombinant lentiviral vector (carrying GFP);
  • Her2 TCR-1B5-wt w/o GFP recombinant lentiviral vector without GFP.
  • Her2 TCR gene fragment was amplified by PCR and cloned into the downstream of the EF1- ⁇ promoter of the two lentiviral vectors (ie, pCDH-EF1 ⁇ -MCS-(PGK-GFP) and pCDH-EF1 ⁇ -MCS): Her2 TCR
  • the TCR fragments of -1B5-dis and TCR-1B5-wt were both 5' primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3' (SEQ ID NO: 28) and 3' primer 5'-GTTGATTGTCGACGCCCTCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 29 ) amplified.
  • the TCR fragment of Her2 TCR-1B5-mC was amplified by 5' primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3' (SEQ ID NO: 30) and 3' primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3' (SEQ ID NO: 31). .
  • the PCR was carried out using a Q5 high-fidelity PCR kit (NEB, cat#M0543S). After the reaction conditions were 98 ° C for 30 seconds, 25 cycles of 98 ° C for 10 seconds, 65 ° C for 10 seconds, and 72 ° C for 3 minutes were performed.
  • the obtained TCR fragment was cloned into the MCS region downstream of the EF1 ⁇ promoter of the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector or the pCDH-EF1 ⁇ -MCS vector.
  • the recombinant TCR lentiviral expression vectors constructed were prepared as described above to obtain respective recombinant TCR lentiviral particles.
  • Her2 TCR-1B5-dis is a disulfide bond added to the TCR constant region by point mutation in the literature "Cancer Res. 2007 Apr 15; 67(8):3898 - 903. The entire disclosure is incorporated herein by reference.
  • Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety by reference. This article.
  • FIG. 3A shows a portion of a T cell line transfected with a lentivirus carrying the Her2TCR-1B5 TCR and GFP genes (i.e., the Her2 TCR-1B5-dis recombinant lentiviral vector and the Her2 TCR-1B5-mC recombinant lentiviral vector).
  • GFP + cells can bind to the Her2-E75 tetramer, indicating that these Lentiviral-transfected T cells recognize the Her2/neu 369-377 polypeptide presented by the HLA-A2 molecule.
  • FIG. 3B shows the exogenous TCR (Her2 TCR) obtained by transfecting J. RT-T3.5 cells expressing only the beta chain or Jurkat cells expressing both the ⁇ chain and the ⁇ chain, and the constant region was replaced by the mouse constant region sequence.
  • the expression level of -1B5-mC) was significantly higher than that of TCR (Her2 TCR-1B5-dis) which added only one disulfide bond in the constant region.
  • FIG. 3C shows that the T cell line expressing exogenous Her2 TCR-1B5 can be activated by T2 cell-expressing Her2/neu 369-377 polypeptide to express CD69, indicating that this TCR has the function of recognizing the Her2/neu 369-377 polypeptide antigen.
  • the expression level of the exogenous TCR (Her2 TCR-1B5-mC) obtained by replacing the constant region with the mouse constant region sequence is not only increased, but also the specific recognition ability for the polypeptide antigen is significantly higher than that of the TCR modified by the additional disulfide bond ( Her2 TCR-1B5-dis) can be activated by lower concentrations of Her2/neu 369-377 polypeptide.
  • the minimum polypeptide concentration for activating Jurkat cells expressing Her2 TCR-1B5-mC was approximately 0.05 ⁇ g/ml, which was approximately 50-fold higher than the polypeptide concentration of the activated Her2 CTL 1B5 clone shown in Figure 1C. This indicates that the ability of the Her2 TCR-1B5 TCR recognition polypeptide antigen expressed by Jurkat cells is significantly lower than that of the Her2 TCR-1B5 TCR expressed on CD8 + CTL.
  • One possible reason is that Jurkat cells do not express CD8 molecules, and CD8 plays an important supporting role in the recognition function of Her2 TCR-1B5.
  • Example 3 Normal peripheral blood T cells are transfected with Her2 TCR-1B5-mC recombinant lentivirus and express a specific TCR that recognizes the Her2/neu 369-377 polypeptide.
  • TCR obtained by the present invention can be expressed in primary T cells and has the function of recognizing the Her2/neu antigen polypeptide
  • recombinant lentiviral particles carrying the Her2 TCR-1B5-mC gene (Her2 TCR-1B5-mC recombination slow)
  • Viral vectors were transfected with peripheral blood T cells from two different normal donors activated by CD3/CD28 antibody, and cells were harvested 7 days later for Her2-E75 tetramer staining. The specific method is as described above. The results are as follows:
  • FIG. 4A shows that both peripheral blood mononuclear cells (#1PBMC and #2 PBMC, respectively) can bind to Her2-E75 tetramer, indicating that Her2 TCR-1B5-mC expressed in these cells can be specific.
  • the Her2/neu antigen polypeptide presented by HLA-A2 is sexually recognized.
  • the results also showed that in the Her2-E75 tetramer-positive cells (ie, expressing Her2 TCR-1B5-mC), most of the positive cells were CD8 + T killer cells, and a small number of positive cells were CD8-like lymphocytes, which is likely CD4 + T helper cells.
  • the fluorescence intensity of CD8 + CTL binding to Her2-E75 tetramer (#2 in Geom.Mean in #2 PBMC samples, 1404 in Geom.Mean in #1PBMC samples) was also significantly greater than CD4 + T cells (#2 PBMC samples)
  • the Geom Mean in the 560 is 560, and the Geom.Mean in the #1PBMC sample is 504).
  • transfection efficiency of lentivirus-infected CD8 + and CD4 + T cells is the same, it indicates that a part of the exogenous Her2/neu 369-377-specific TCR expressed on CD4 + cells does not bind to Her2-E75 tetramer, even if bound, Affinity is also lower than exogenously transfected TCR expressed on CD8 + T cells. This further demonstrates that the transfected TCR requires the auxiliary function of the CD8 molecule to effectively bind the Her2/HLA-A2 complex.
  • TCR-transfected PBMC cells were added to each well of a 96-well plate, and Her2/neu 369-377 antigenic peptide (Her2/neu 369-377 antigen) was presented at different concentrations in T2 cells (10e5 per well).
  • the polypeptide was 10-fold diluted from 0.1 ⁇ g/ml to obtain a final concentration of 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, and 0.001 ⁇ g/ml.
  • the IFN- ⁇ secreted by T cells in the supernatant was detected. To determine the function of this TCR-expressing PBMC cell to specifically recognize the Her2/neu 369-377 polypeptide.
  • the target cells of the control group were T2 cells which presented 1 ⁇ g/ml of the EBV virus antigen polypeptide LMP2 426-434 which can bind to the HLA-A2 molecule.
  • Figure 4B shows that PBMC expressing Her2 TCR-1B5-mC can be activated by T2 cell-derived Her2/neu 369-377 antigen polypeptide to secrete IFN- ⁇ , indicating the expression of exogenous Her2 TCR-1B5-mC primary T
  • the cells can specifically recognize the Her2/neu 369-377 polypeptide presented by the HLA-A2 molecule.
  • the ability to recognize antigenic polypeptides correlates with the amount of exogenous TCR expressed on T cells.
  • FIG. 4C shows that T cell secreting IFN- ⁇ is significantly inhibited by the addition of anti-human CD8 antibody when T cells are co-cultured with T2 cell-presenting antigen polypeptide (T2+Her2-E75, ie Her2/neu 369-377 polypeptide). .
  • T2+Her2-E75 T2 cell-presenting antigen polypeptide
  • Her2/neu 369-377 polypeptide T2 cell-presenting antigen polypeptide
  • Example 4 Her2/neu 369-377 polypeptide-specific TCR expressed by normal peripheral blood T cells transfected with Her2 TCR-1B5-mC recombinant lentivirus recognizes HLA-A2 + Her2/neu + tumor cells
  • 10e5 PBMCs transfected with Her2 TCR-1B5-mC w/o GFP recombinant lentivirus were added to each well of a 96-well plate (shown as #2 PBMC transfected with Her2 TCR-1B5), or 10e5 control #2 PBMCs that were not transfected with TCR were mixed with 10e5 different tumor cell lines, and then the secreted IFN- ⁇ was detected in the supernatant.
  • the specific method is as described above. The results are as follows:
  • FIG. 5A shows that T cells expressing Her2 TCR-1B5-mC can be activated by HLA-A2 + Her2/neu + tumor cell lines and secrete IFN- ⁇ .
  • Tumor cell lines include colon cancer Colo205 cells, breast cancer MDA- MB-231 cells, colon cancer Caco-2 cells, and lung cancer H1355 cells.
  • HLA-A2 - Her2/neu + ovarian cancer SK-OV3 cells, lung cancer H647 cells, and HLA-A2 + Her2/neu - lymphoma Bjab cells do not activate T cells transfected with Her2 TCR-1B5-mC TCR .
  • Her2 TCR-1B5-mC TCR can specifically recognize the Her2/neu antigen presented by HLA-A2 on the surface of tumor cells.
  • Control T cells from the same donor PBMC that were cultured in parallel but not transfected with Her2 TCR-1B5-mC were not activated by the listed tumor cell lines, indicating that the response to tumor cells was not non-specific.
  • Figure 5B shows that T cells expressing Her2 TCR-1B5-mC TCR (#2 PBMC transfected with Her2 TCR-1B5) showed significant responses to colo205 cells, and recognition functions were enhanced by anti-CD8 antibodies and anti-HLA molecules.
  • the antibody is almost completely blocked, which further indicates that the effector cells that recognize the Her2/neu antigen on the surface of tumor cells are CD8 + killer T cells, and their specific antigen recognition function depends on the auxiliary function of CD8, which is also related to the recognition of T2 cells.
  • the Her2/neu 369-377 polypeptide antigen was consistent in Her2 TCR-1B5-mC TCR.
  • the difference in sensitivity of different tumor cell lines to specific T cells may be related to the expression of different levels of Her2/neu antigen polypeptide/HLA-A2 complex in tumor cells, and may also be related to the inhibition of T cell function by tumor cells themselves.
  • the high-affinity TCR that specifically recognizes the Her2/neu 369-377 polypeptide can be obtained by in vitro induction of the Her2/neu 369-377 polypeptide, these high-affinity TCRs often fail to recognize the Her2/neu presented by the tumor cells.
  • Antigen Cancer Res. 1998; 58: 4902 - 4908. Cancer Immunol. Immunother. 2008; 57: 271 - 280).
  • Her2/neu 369-377 polypeptide binding HLA-A2 molecule differs from the configuration of the polypeptide/HLA complex presented in the cell (see the journal “Journal of Immunology, 2008, 180". :8135–8145”).
  • the Her2/neu 369-377 polypeptide acts as a mimotope antigen, and the specific TCR induced can recognize both Her2/neu 369-377 polypeptide and similar polypeptides presented by tumor cells.
  • the Her2/neu 373-382 polypeptide see the literature "J Immunol.
  • TCR Her2/neu 369-
  • the 377 polypeptide has high affinity but does not effectively recognize the corresponding mimotope polypeptide presented by the tumor cell.
  • the TCR specifically recognizing the Her2/neu 369-377 polypeptide of the present invention not only targets the Her2/neu 369-377 polypeptide proposed by the tumor cell, but also recognizes the Her2/ derived from the tumor cell.
  • Other mimotope polypeptides of neu are also recognized.
  • TCRs that naturally exist in the peripheral T cell pool that recognize the Her2/neu antigen are mostly low-intensity.
  • Another high-affinity TCR that recognizes CD8 function-independent high-affinity TCRs from tumor cells is paired by multiple alpha and beta chains of the Her2/neu 373-382 polypeptide-specific T cell population. (See the literature "HUMAN GENE THERAPY 2024, 25: 730-739"; WO/2017/133779). Since it is not directly obtained from specific monoclonal T cells, it is not possible to determine whether this TCR is present in the peripheral natural T cell pool.
  • TCRs high-affinity TCRs
  • function of these TCRs does not depend on the helper function of CD8, and thus the complementation of CD8 + killer T cell function can be obtained by transfecting CD4 + T cells.
  • CD8 function-dependent TCR the same purpose can be achieved by simultaneously expressing the TCR and the exogenous CD8 molecule by the expression vector.
  • the present invention provides a Her2/neu 373-382 polypeptide-specific TCR ⁇ chain and ⁇ chain full sequence induced from an in vitro pericellular T cell bank of HLA-A2 + , which is expressed and expressed after transfection.
  • Primary killer T cells of the region-modified TCR can recognize a variety of HLA-A2 + Her2/neu + tumor cells.
  • New methods and approaches have been provided for the development and clinical application of adoptive transfer of specific TCR-modified T cells to treat tumors.

Abstract

L'invention concerne un récepteur de lymphocytes T isolé, une cellule modifiée par celui-ci, des acides nucléiques codants, un vecteur d'expression, un procédé de préparation, une composition pharmaceutique et des applications. Le récepteur de lymphocytes T (TCR) isolé comprend au moins l'une d'une chaîne alpha et une chaîne bêta, à la fois la chaîne alpha et la chaîne bêta, comprenant une région variable et une région constante, et est caractérisé en ce que le récepteur de lymphocytes T identifie de manière spécifique l'antigène Her2/neu exprimé par des cellules tumorales, la séquence d'acides aminés de la région variable de la chaîne alpha a une cohérence d'au moins 98 % avec la séquence d'acides aminés telle que représentée par SEQ ID NO : 19, et la séquence d'acides aminés de la région variable de la chaîne bêta a une cohérence d'au moins 98 % avec la séquence d'acides aminés telle que représentée par SEQ ID NO : 20. Le TCR identifie de manière spécifique l'antigène tumoral et, en même temps, empêche toute réaction possible de toxicité hors cible. L'utilisation du TCR, selon l'invention pour modifier une cellule immunitaire fournit des effets antitumoraux significatifs.
PCT/CN2018/115191 2017-11-14 2018-11-13 Récepteur de lymphocytes t isolé, cellule modifiée par celui-ci, acides nucléiques codants, vecteur d'expression, procédé de préparation, composition pharmaceutique et applications WO2019096115A1 (fr)

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